Contraction of cardiac ventricular myocytes is initiated by Ca influx (ICa) during the action potential, which activates Ca release via ryanodine receptors (RyRs) from junctional sarcoplasmic reticulum (SR). The majority of Ca influx and release occurs at invaginations of the surface membrane, the t-tubules. However, t-tubule structure is disrupted during heart failure, leading to increased spatiotemporal heterogeneity of Ca release, although it is unclear whether the function of the remaining t-tubules is also altered. We have therefore investigated ICa density in the t-tubules and surface membrane of ventricular myocytes isolated from failing rat hearts.Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Coronary artery ligation (CAL) was performed in male Wistar rats (200-250g) under anaesthesia (ketamine 75 mg/kg, medetomidine 0.5 mg/kg, i.p.) with appropriate analgesia (buprenorphine 0.05 mg/kg s.c.). 17 weeks post-surgery, hearts were excised from ligated (CAL) or sham-operated (Sham) animals under pentobarbitone (140 mg/kg i.p.) anaesthesia and left ventricular myocytes isolated by enzymatic digestion. ICa was recorded using whole-cell patch-clamp in intact cells and following acute detubulation (~25°C). Cells were stained with di-8-ANEPPS to determine t-tubule structure, and RyRs labelled using standard immunolabelling techniques, before imaging by confocal microscopy. Intracellular Ca was monitored using Fluo-4 by confocal line scan imaging. T-tubule regularity was quantified using FFT analysis. Data are expressed as mean±SEM (n cells). Student’s t-test or ANOVA were used for statistical analysis, with the appropriate Bonferroni post hoc test; the confidence level was p<0.05.Animals that underwent CAL had significantly increased heart:body weight and lung:body weight ratios. Myocytes isolated from CAL animals had increased cell capacitance, disrupted t-tubule structure and decreased t-tubule regularity, although RyR distribution appeared unchanged. They also exhibited a smaller systolic Ca transient, with slower time to peak, compared to Sham. ICa density was not different in intact myocytes from Sham and CAL animals (Sham -5.98±0.23 (37), CAL -5.80±0.26 (33), pA/pF, ns). However, following detubulation ICa density was smaller in Sham than in CAL myocytes (Sham -4.03±0.17 (28), CAL -5.34±0.27 (31), pA/pF, p<0.001). These data suggest that the distribution of ICa changes in myocytes from CAL animals, with a decrease in ICa density at the t-tubules (by ~30%, p<0.001) and an increase at the surface membrane (by ~150%, p<0.001), so that the t-tubule:surface ICa ratio decreases from ~7 to ~2. In the absence of associated changes in RyR distribution, the loss of ICa from the t-tubules may contribute to the impaired excitation-contraction coupling observed in HF.